Targeting Axl and Mer Kinases in...

Review

Targeting Axl and Mer Kinases in Cancer

Anupam Verma1,2, Steven L. Warner2, Hariprasad Vankayalapati2, David J. Bearss2, and Sunil Sharma2

AbstractReceptor tyrosine kinases (RTK) are cell-surface transmembrane receptors that contain regulated kinase

activity within their cytoplasmic domain and play an important role in signal transduction in both normal and

malignant cells. The mammalian TAM RTK family includes 3 closely related members: Tyro-3, Axl, and Mer.

Overexpression or ectopic expression of the TAM receptors has been detected in a wide array of human

cancers. Growth arrest-specific gene 6 has been identified as the major ligand for these TAM RTKs, and its

binding to the receptors has been shown to promote proliferation and survival of cancer cells in vitro.

Abnormal expression and activation of Axl or Mer can provide a survival advantage for certain cancer cells.

Inhibition of Axl andMermay enhance the sensitivity of cancer cells to cytotoxic agents andwould potentially

be a therapeutic strategy to target cancer cells. This review elucidates the role of Axl and Mer in normal

cellular function and their role in oncogenesis. In addition, we review the potential to inhibit these RTKs for

the development of therapeutic targets in treatment of cancer.Mol Cancer Ther; 10(10); 1763–73.�2011 AACR.

Introduction

Receptor tyrosine kinases (RTK) are a large family oftransmembrane proteins exhibiting great diversity intheir extracellular regions, although sharing in commona highly conserved intracellular tyrosine kinase domain.They function as sensors for extracellular ligands, thebinding of which triggers receptor dimerization andactivation of the receptor’s kinase activity. This activationleads to the recruitment, phosphorylation, and activationof multiple downstream signaling proteins, which ulti-mately change the physiology of the cell. RTKs regulatecellular processes, including survival, growth, differen-tiation, adhesion, proliferation, and motility. Fifty-eightknown RTKs in the human genome are classified into 20families by amino acid sequence identity within thekinase domain and structural similarities within theirextracellular regions. One subfamily is referred to asthe TAM family, identified in 1991, comprising Tyro-3(also called Sky), Axl, and Mer. The TAM receptors arecharacterized by a combination of 2 immunoglobin-likedomains and dual fibronectin type III repeats in theextracellular region and a cytoplasmic kinase domain(Fig. 1A). The primary ligand for TAM receptors isgrowth arrest-specific 6 (Gas 6), a fairly large (75 kDa)

vitamin K–dependent protein known to activate down-stream signaling (1).

Axl also called Ark and Ufo, was originally detectedin 1988 from 2 patients with chronic myelogenousleukemia (CML) as an unidentified transforming gene.Axl was later cloned from patients with CML andchronic myeloproliferative disorders (2). The nameAxl is derived from the Greek word anexelekto, whichmeans "uncontrolled." The human axl gene is located onchromosome 19q13.2 and encodes 20 exons (Fig. 1A; ref.2). Axl is ubiquitously expressed and has been detectedin a wide variety of organs and cells, includingthe hippocampus and cerebellum, monocytes, macro-phages, platelets, endothelial cells (EC), heart, skeletalmuscle, liver, kidney, and testis (3–5). Subsequent to itsoriginal identification in CML, Axl overexpression hasbeen reported in several human cancers including colon(6), esophageal (7), thyroid (8), breast (9), lung (10), liver(11), and astrocytoma-glioblastoma (12).

The second member of the TAM family was isolatedfrom the chicken retrovirus RLP30 and was named v-rykand later cloned from embryonic chicken brain andrenamed to c-eyk (13). It was later named c-Mer afterbeing cloned from the human B-lymphoblastoid expres-sion library, as it was found in monocytes, epithelial, andreproductive tissues (4). It is also called MerTK andmapped to chromosome 2q14.1 and contains 19 exons(Fig. 1A; ref. 14). Mer is expressed in hematopoieticlineages such asmonocytes, macrophages, dendritic cells,natural killer (NK) cells, megakaryocytes, and platelets(3, 4). Since its original detection in B- and T-cell leuke-mias (15), it has also been detected in mantle celllymphomas (16), gastric cancer (17), pituitary adenomas(18), melanoma (19), prostate cancer (20), and breastcancer (9, 21, 22).

Authors' Affiliations: 1Pediatric Hematology/Oncology, Primary Chil-dren's Medical Center, and 2Center for Investigational Therapeutics,Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah

Corresponding Author: Anupam Verma, Huntsman Cancer Institute,2000 Circle of Hope, Salt Lake City, UT 84112. Phone: 801-587-5559;Fax: 801-585-0101; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-11-0116

�2011 American Association for Cancer Research.

MolecularCancer

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Published OnlineFirst September 20, 2011; DOI: 10.1158/1535-7163.MCT-11-0116

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Figure 1. A, DNA structure ofaxl and mer aligned with theirfunctional protein domains (top 2diagrams). Human axl geneencodes 20 exons, whereas merencodes 19 exons. Domainstructure of Gas 6, ligand for Axland Mer (bottom). B, TAMreceptors and their ligands alongwith signaling pathways for Axland Mer. Ig, immunoglobulin;FNIII, fibronectin type III; GLA,gamma-carboxyglutamic acid.

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The biological ligands for Axl and Mer are 2 highlysimilar vitamin K–dependent proteins, Gas 6 and proteinS (Fig. 1A). Both proteins have an N-terminal regioncontaining a modified g-carboxyglutamic acid residue(G1a), which has the ability to interact with negativelychargedmembrane phospholipids tomediate the bindingof both Gas 6 and protein S to apoptotic cells. The G1adomain mediates Ca2þ-dependent binding to negativelycharged membrane phospholipids exposed on thesurface of apoptotic cells. The G1a domain is followedby a loop region, 4 epidermal growth factor (EGF)–likerepeats, and 2 C-terminal globular laminin G (LG)–likedomains, which house a globular sex hormone bindingglobulin (SHBG)–like region. This SHBG domain bindsdirectly to and activates Axl, and a supporting role hasbeen implicated for the G1a region in the function of Gas 6(23, 24). Gas 6 has 3- to 10-fold lower affinity for Mer thanAxl (23). Studies using cultured cell lines have shown anAxl-mediated effect of exogenous Gas 6 on cell survival,proliferation, migration, and adhesion (25–28). Currently,no studies show any connection between protein S andactivatedAxl, though it was recently shown that protein Scan bind and activate endogenous murine Mer. Recentdata suggest tubby and Tulp1 as novel bridging mole-cules to facilitate phagocytosis through Mer (29).

Axl and Mer Signaling Pathways

Fms-Mer receptor chimera and EGF-Axl receptorchimera studies were the first to elucidate the TAMreceptor signaling pathways. Signaling pathways down-stream from the Mer and Axl kinases include growthfactor–mediated proteins such as phosphoinositide3-kinase (PI3K), RAt sarcoma (RAS), and extracellularsignal regulated kinase (ERK; Fig. 1B).Studies using chimeric Mer receptors expressed in

NIH3T3 fibroblasts linked downstream signaling path-ways, such as PI3K, phospholipase C-g (PLCg), and ERK,to Mer activation. Gas 6–dependent activation of Merstimulates phosphorylation of ERK1/2, leading to cellu-lar transformation and increased proliferation and DNAsynthesis. The ultimate downstream targets of the path-way differ according to cell type and tissue microenvi-ronment. In leukemia cells, ligand-dependent activationof EGF receptor (EGFR)–Mer chimeric receptor stimu-lates phosphorylation of Akt, ERK 1/2, and p38 mitogen-activated protein kinases (MAPK), which results indecreased apoptosis but no change in proliferation(30). Expression of CD8-Mer chimera in pro-B cells resultsin transcriptional activation of NF-kB via PI3K/Akt.Additional activation of p38/MAPK andmeiosis-specificserine/threonine protein kinase 1 (MEK1) occurs viaCD8-Mer, leading to protection from apoptosis. Someatypical signaling pathways involved in cell survivalhave been studied as a link between Mer and theactin cytoskeleton via growth factor receptor-boundprotein 2 (Grb2), Shc, and Vav1. Downregulation of theproapoptotic tumor suppressor WW domain-containing

oxidoreductase (Wwox) may be a mechanism by whichactivated Cdc42-associated kinase 1 (Ack1) andMer relaysurvival signals in cancer cells (31).

An early study that screened an expression libraryrevealed p85a and p85b subunits of PI3K and PLCg asbinding partners for the Axl intracellular domain (32).Gas 6/Axl signaling promotes growth, survival, andproliferation of numerous cell types by activation ofthe RAS/RAF/MAPK/ERK1/2 and PI3K signaling path-ways (23, 33). The RAS/ERK pathway is essential for Gas6–induced mitogenesis of NIH3T3 cells, which may beactivated by multiple TAM receptors. The MAPK/ERKpathway results in Axl-mediated proliferation and Axlbinding to and activation of PI3K linked to multipledownstream pathways, leading to increased cell survival.One such pathway is the classical PI3K stimulation of Aktand S6 kinase (S6K; ref. 25). Downstream survival path-ways activated by Gas 6/Axl signaling via PI3K/Akt alsoinclude phosphorylation of NF-kB, increased expressionof antiapoptotic proteins such as B-cell lymphoma gene2 (Bcl-2) and B-cell lymphoma extra large (Bcl-xL), andinhibition of proapoptotic proteins such as caspase 3 (34).Suppressor of cytokine signaling (SOCS-1) was originallyidentified as a negative regulator of cytokine signalingand could serve a similar role in Axl signaling. Thenoncatalytic region of tyrosine kinase adaptor protein 2(Nck2) is an adapter protein composed of 3 tandem SH3domains and 1 SH2 domain, which may serve to tetherAxl to other signaling complexes (35). The Axl-Nck2interaction connects Axl to a ternary complex consistingof the particularly interesting new cysteine-histidine–richprotein (PINCH) and integrin-linked kinase (ILK),which is a signaling platform at focal adhesions regulat-ing cytoskeleton dynamics and downstream signalingpathways. Gas 6/Axl signaling has also been linkedto neuronal cell migration. Studies of GnRH neuronssuggest that Axl directs migration of these cells via asignaling pathway involving PI3K, RAS, RAC, p38MAPK, MAPKAP kinase 2, and HSP25, resulting in actinreorganization (36).

Soluble Axl and Mer

Membrane-bound receptors generate soluble ligand-binding domains either by proteolytic cleavage of theextracellular domain or alternative mRNA splicing,yielding a secreted protein (Fig. 1B). Axl exists as atransmembrane protein and as a soluble molecule, whichwas recently shown in mouse serum (37). Constitutiveand phorbol 12-myristate 13-acetate–induced generationof soluble Axl (sAxl) involves the activity of disintegrin-like metalloproteinase 10 (ADAM10). Spontaneousand inducible Axl cleavage is inhibited by the broad-spectrum metalloproteinase inhibitor GM6001 and byhydroxamate GW280264� (Fig. 2), which is capable ofblocking ADAM10. Experimental evidence exists thatsupports a role for immobilized sAxl in promoting cellmigration and activation of membrane-bound full-length

Axl and Mer in Cancer

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Axl and its downstream target, PI3K. A dynamic equi-librium between sAxl and Gas 6 levels in biologicalfluids may have an important regulatory role and affectGas 6 function. sAxl may increase the bioavailability of

Gas 6 by prolonging its half-life and slowing ligandrelease, thereby resulting in local or systemic effects ofthis protein and the nature and/or duration of thesignaling event. A potential ability of sAxl to serve

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Figure 2. A, binding mode of a small molecule inhibitor in complex with Axl kinase. The surface represents the active site residues, and the spheresdepict the hydrophobic and gatekeeper site residue interactions. B, chemical structures of reported Axl and Mer small molecule inhibitors. The structures ofGM-6001 and GW-280264�, two protease (ADAM 10) inhibitors.

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as a natural antagonist of Gas 6 could have clinicalrelevance. Similarly, the membrane-bound Mer proteinis cleaved in the extracellular domain via a metallo-proteinase (38). Further studies are needed to establishsAxl and sMer as important biomarkers for correlationwith disease stage and predicting prognosis.

Role of TAM Receptors in Cancer

Proto-oncogenes can be activated by a variety ofmechanisms, including gene amplification and muta-tions, proteolytic cleavage, and altered protein expres-sion. To date, no activating TAM receptor mutationshave been associated with the development of cancer;however, aberrant regulation of these signaling path-ways and cellular processes play an important role inoncogenic transformation. Therefore, overexpressionand ligand-induced activation represent the primarymechanisms of activation in a wide array of humancancers (Table 1).TAM receptors activate prosurvival signaling path-

ways in both normal and cancer cells. In some cases,they prevent apoptosis without stimulating prolifera-tion (30), whereas in others they increase proliferationwithout inhibiting apoptosis (26). Finally, some TAMreceptors simultaneously promote both survival andproliferation. An example of Mer-mediated cell survivalis by activation of the Ack1 and downregulation ofthe tumor suppressor Wwox, as discussed above.Expression of the constitutively active Ack1 in humanprostate adenocarcinoma cells induced anchorage-independent growth and increased tumor growth in

an ectopic xenograft model (31). A 4- to 5-fold increasein phosphorylated Ack1 and 6-fold decrease in Wwoxproteins were detected in patients with advanced stageprostate cancer compared with normal prostate. Mer isnot expressed in normal human lymphocytes, but isectopically expressed in T-cell leukemias and E2A-PBX1–positive B-cell leukemias (39, 40). Lymphocytesfrom a Mer transgenic mouse model exhibited a func-tional survival advantage in vitro compared with wild-type lymphocytes when treated with glucocorticoids, astandard leukemia therapy.

TAM receptor signaling pathways have been impli-cated in the regulation of the actin cytoskeleton, whichresults in changes in cellular morphology. Axl is over-expressed in glioblastoma cells, and an experiment witha dominant negative form of Axl resulted in reducedmotility, altered morphology with loss of filopodia,and loss of cell-to-cell interactions, eliciting its rolein oncogenesis (12). In breast cancer models, ectopicexpression of Axl conferred a highly invasive pheno-type to weakly invasive MCF7 cells. Inhibition of Axlsignaling by short hairpin (shRNA) knockdown or ananti-Axl antibody decreased the motility and invasive-ness of highly invasive breast cancer cells (41).

The extracellular domains of the TAM receptors con-tain adhesion molecule–like motifs and may be involvedin cell–cell contacts. Overexpression of murine Axl hasbeen shown to result in cell aggregation. Experimentshave shown that Axl-expressing cells bind to immobi-lized Axl extracellular domain, mediated by hemophilicbinding. Studies have shown that cell aggregation is theresult of a Gas 6–mediated interaction between Axl and

Table 1. Upregulation of Axl/Mer/Gas 6 in cancer

Number Cancer type Upregulation of Axl/Mer/Gas 6 Reference

1 Acute leukemia (ALL, AML) Axl, Mer (15, 39, 40, 49, 60, 68)2 Astrocytoma Axl, Mer, Gas 6 (12, 51, 53)3 Breast cancer Axl, Mer, Gas 6 (9, 22, 54–56, 58)4 Colorectal carcinoma Axl (6)5 Esophageal adenocarcinoma Axl (7, 69)6 Gastrointestinal stromal tumors Axl (70)7 Gastric cancer Axl, Mer, Gas 6 (71)8 Hepatocellular carcinoma Axl (11, 61)9 Kaposi sarcoma Axl (72)10 Lung cancer Axl (10)11 Mantle cell lymphoma Mer (16)12 Melanoma Axl, Mer (19)13 Ovarian cancer Axl, Gas 6 (67)14 Osteosarcoma Axl (44)15 Pancreatic ductal adenocarcinoma Axl, Gas 6 (46, 73)16 Renal cell carcinoma Axl, Gas 6 (74)17 Prostate cancer Axl, Mer (20, 26, 31)18 Thyroid cancer Axl, Gas 6 (8)19 Uterine endometrial cancer Axl, Gas 6 (75)






neighboring cells. Axl expression correlates with theadherence of lung cancer cells (42). sAxl bound to theextracellular matrix may constitute a chemoattractant forAxl-mediated migration, as scratch tests revealed thatimmobilized Axl-Fc promotes migration of primaryfibroblasts prepared from Axl wild-type mice (37).

Angiogenesis is the formation of new blood vesselsand commonly promotes tumor growth and transfor-mation. Proliferation and migration of vascular smoothmuscle cells (VSMC) are required for angiogenesis.VSMCs express Gas 6, and exogenous application ofGas 6 promotes proliferation and migration of VSMCs(27). Genetic silencing of Axl or Gas 6 significantlyreduced migration of human umbilical vein endothelialcells (HUVEC), whereas overexpression of Axlincreased HUVEC growth and tube formation (43).Axl knockdown in HUVECs led to upregulation ofAng-2, the angiopoietin signaling system that plays akey role in the regulation of angiogenesis, vascularhomeostasis, and vascular regression. Ang-1 is anagonist that supports EC survival and endotheliumintegrity through the PI3K/Akt signaling pathway. Inthe presence of VEGF, Ang-2 provides an importantangiogenic stimulus. Thus, Axl knockdown is additivewith anti-VEGF agents in inhibiting endothelial tubeformation (12).

Axl kinase activity is important for the regulation ofEC growth and regulates tube formation in a kinase-independent manner. Elevated Axl correlates with ad-herence, motility, and invasiveness of osteosarcoma celllines selected for their high metastatic ability in an in vivomodel of lung metastasis (44). Axl expression also corre-lates with invasiveness of lung cancer lines in vitro and inpatients with adenocarcinoma (10). In a recent study, anorthotopic breast cancer model was used to investigatethe functional significance of Axl in metastasis, in whichAxl expression was required for MDA-MB-231 cells toestablish metastatic foci in the lung. Axl knockdown byshRNA completely abolished the ability of cells emergingfrom the primary tumors to colonize the lungs. This studyimplicated Axl in the early stages of the MDA-MB-231cancer cell metastasis and provided the first in vivoevidence that directly links Axl to metastasis (45).Recently, an experiment showed that downregulationof numerous transcripts associatedwith the phenomenonof cancer cell invasion, including significant reduction inlevels of snail, slug, and twist mRNA, whose productsregulate the process of epithelial-mesenchymal-transition(EMT; ref. 46). EMT is required for vascular intravasationandmetastatic seedingby tumor cells, and the snail/slug/twist family of transcriptional repressors plays an impor-tant role in phenotypic switch. This group is the firstto describe an association between transcription factorsfacilitating EMT andAxl and, hence,mechanistic basis forthe observedblockade in invasion and/ormigrationuponknockdown of this RTK in MIA PaCa-2 cells.

Roles for Axl or Mer kinases have been reported in avariety of tumor types and have been extensively

reviewed elsewhere (47). This review focuses on thecancers reported to be the most closely associated withAxl and Mer function. In addition to playing a role incancer initiation and progression, Axl andMer have beenstrongly associated with other disease physiology, suchas the immune system and autoimmune disease. Thistopic has also been recently reviewed in detail (48).

Axl in Pancreatic Carcinoma

Ductal adenocarcinoma of the pancreas is a uniformlylethal malignancy. Immunohistochemical (IHC) analysisfor Axl expression was done on a panel of 99 pancreaticcancers using anti-Axl–specific antibody, and Axl label-ing was observed in 55% of cases (46). Axl proteinexpression was significantly associated with lymph nodemetastasis, and patients harboring Axl-positive tumorshad a significantly shorter median survival of 12 months,compared with Axl-negative cancers of 18 months. Thisfinding was reaffirmed by the same group inMIA PaCa-2pancreatic cancer cells with genetic knockdown ofendogenous Axl protein, which resulted in profound lossof invasive and migratory capabilities, accompanied bynear-complete loss of filopodial extensions. Axl promotesthe growth and survival of neoplastic and nonneoplasticcells through activation of MAPK and the PI3K/Aktsignaling pathways, and both effector arms are inhibitedin MIA PaCa-2 cells upon Axl knockdown, as evidencedby the decreased phosphorylation of the ERK1/2, PDK1,and Akt (33, 49). Recently, an association between tran-scription factors facilitating EMT and Axl and, hence, amechanistic basis for the observed blockade in invasionand/or migration upon knockdown of this RTK in MIAPaCa-2 cells has been described. Matrix metalloprotei-nase-9 (MMP-9), a type IV collagenase involved in base-ment membrane proteolysis and in tumor angiogenesis,is expressed in the tumor microenvironment promotinginvasion and metastasis. MMP-9 is reported to be over-expressed in pancreatic cancers, and recently, it has beendocumented that Axl in cancer cells can upregulateMMP-9 and render the cells more invasive (50).

Axl, Gas 6, and Mer in Human Gliomas

Studies of fresh-frozen tumor samples and IHC studiesof paraffin-embedded tumor tissue showed that Axl andGas 6 are frequently overexpressed in World HealthOrganization (WHO) grade 2 and 4 gliomas, includingthose of astrocytic, oligodendroglial, and ependymalorigins (51). Glial fibrillary acidic protein (GFAP), anastrocyte-specific intermediate filament controlling cellshape and cell movement, is commonly detectable inglioma cells. In these studies, vessels typicallysurrounded by Axl and GFAP-positive tumor cells repre-sented necrotic areas and pseudopalisades caused byintravascular thrombosis. Both Axl and Gas 6 werestrongly expressed in ECs of microvascular hyperplasia.Tumor cells adjacent to microvascular hyperplasia

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showed a pronounced Axl staining. A comparative IHCstudy showed that Axl and GFAP are coexpressed inglioma cells of pseudopalisades, thus showing a func-tional association of Axl and GFAP during glioma cellmigration. Inhibition of Axl signaling almost completelysuppressed glioma cell migration into fetal rat brainspheroids and into brain tissue of a mouse xenograftmodel (12). Cell death due to apoptosis and coagulativenecrosis is fundamental to glioblastoma multiforme(GBM), and studies have shown Gas 6/Axl signalingto attenuate neuronal cell death caused by serum star-vation, secretory phospholipase A2, and amyloid b pro-tein (52). A strong coexpression of Axl/Gas 6 is found inreactive astrocytes and astrocytic end feet, which are inclose proximity to micro-vessel walls and modulatethe blood–brain barrier function. Axl may also have acomparable function in modulating tumor angiogenesisand blood–brain barrier function similar to VEGF andplatelet-derived growth factor receptors. Recently, Merand Axl overexpression has been shown in a majority ofGBM cell lines and patient samples. Recent findingsalso show that inhibition of Mer significantly reducesmigration of GBM cells in vitro (53).

Axl in Breast Cancer

Axl is overexpressed in human breast cancer cell linesand patient samples and correlates with advancedtumor stage (9, 54). Axl and Mer are both upregulatedin metastasis relative to primary tumors and are asso-ciated with poor survival (22, 55). Axl expression isfound in estrogen receptor–positive patients, whereasGas 6 is upregulated in progesterone-positive patients.Overexpression of Axl correlates with poor prognosis,and upregulation of Gas 6 mRNA levels correlates withfavorable prognosis (54, 56). Studies have shown Axlinhibition reduces migration and invasion of breastcancer cells by Axl-dependent expression of MMP-9(50). Recent studies have shown a role for Axl inEMT-associated transcription factors twist, snail, andslug and metastasis of breast cancer. Murine breasttumors treated with an Axl inhibitor exhibit reducedsnail expression in vivo (57). There is evidence that Axlalso has a role in chemoresistance development, as shownwhen chronic exposure of an Axl-negative breast cancercell line to lapatinib resulted in lapatinib resistance, de novoexpression of Axl, and increased expression of estrogenreceptor (58). Lapatinib sensitivity was restored by inhi-bition of either Axl or estrogen receptors.

Axl and Mer in Non–Small Cell Lung Cancer

High levels of Axl, Mer, and their ligands Gas 6 andprotein S have been found inmore than 50% of non–smallcell lung cancer (NSCLC) cell lines (42, 47). Axl over-expression was also shown in 48.3% of patient samples oflung adenocarcinoma and correlated with lymph nodemetastasis and higher clinical stage, indicating it to be a

poor prognostic factor (10). RNA interference (RNAi)–mediated silencing of Axl reduces the viability of NSCLCcells in vitro and inhibits tumor growth in xenograftmodels (45). Axl expression correlates with NSCLC cellinvasiveness and migration, an effect that is mediated byAxl-dependent upregulation of MMP-9 expression (50).

Axl and Mer in Acute Leukemia

One common chromosomal rearrangement found inpre-B–acute lymphoblastic leukemia (ALL) is the t(1:19),resulting in fusion of 2 transcription factors, E2A andPBX1. Historically, E2A-PBX1 positivity is a poor prog-nostic indicator (59). Survival rates for children withrelapsed ALL remain poor with contemporary therapyprotocols. A recent study showed that Mer expression isassociated with poor outcome in the E2A-PBX1 cyto-genetic subgroup in newly diagnosed pre-B–ALL andalso in various cytogenetic subtypes in chemoresistant orrecurrent pre-B–ALL (40). Previous studies had identifiedERK1/2 and Akt as downstream targets of Mer, and arecent study showed a link between Mer and mTORsignaling. ERK1/2 protects cancer cells from apoptosis,and in adults with ALL, it is associated with poor prog-nosis. In AML, Axl overexpression correlates with worseprogression-free and overall survival (60).

Axl in Hepatocellular Carcinoma

A recent study using hepatocellular carcinoma (HCC)cells and cDNAmicroarray showed a 4.7-fold increase ofAxl mRNA levels in Hca-F cells with high lymphaticmetastasis potential, as compared with Hca-F cells withlow lymphatic metastasis potential (61). The group alsofound that Hca-F cells with higher Axl levels had aproliferative advantage. siRNAs were used to inhibitAxl expression in Hca-F cells and showed that the silenc-ing of Axl could impede Hca-F cell proliferationand anchorage-independent growth and resulted in de-creased capacity to migrate and loss of capacity to invadethe surrounding matrix in vitro. The study also showedthat the angiogenic factor Cyr61, which belongs to afamily of CCN proteins and promotes cell adhesion,proliferation, migration, and angiogenesis through cell-type–specific binding to different integrin receptors, wasthe principle gene affected by Gas 6 stimulation of Axl inthe Hca-F cells. Elevated levels of Cry61 correlated withpoor prognosis, lymph-node involvement metastasis,and mortality and can be modulated by Gas 6. Morestudies need to be done to more fully show the role of theAxl/Gas 6 signaling pathway and any role for Mer inHCC.

Axl and Mer as Molecular Therapeutic Targets

Drug designAxl/Gas 6 and Mer signaling pathways represent

novel biologic targets and could interact synergistically






with standard chemotherapeutic agents, permitting dosereduction and toxicity. A crystal structure of Mer wasrecently published in complex with an ATP-competitiveinhibitor (62), and this structure has allowed the con-struction of predictive homology models of Axl. Inaddition to the overall conserved structure of thekinase domain, the ATP-binding pockets of Axl andMer possess features and establish molecular interac-tions with ATP that are common to other proteinkinases. Understanding these interactions has provenuseful in the design and identification of Axl and MerATP-competitive inhibitors (Fig. 2A).

In one example, the crystal structure of Mer wasdetermined in complex with a small molecule ATP-com-petitive inhibitor (compound-52), on the basis of atrisubstituted purine scaffold (Fig. 2B). The cocrystalstructure of the complex revealed the compound occu-pying the adenine and sugar pockets of Mer andmaking important hydrogen bonds, hydrophobic inter-actions, and van der Waals contacts with criticalamino acid residues. The binding mode analysis of theMer-compound-52 complex structure indicated that thecompound binds within the ATP-binding pocket in sucha way that the pyrimidine ring –N1 and –NH of aryl ringat 2-position are involved in hydrogen-bonding acceptorand/or donor interactions with hinge residue Met674backbone –NH. . .N– and –C¼O. . .HN– atoms. The sub-stituent at the fifth position is involved in nonbondedinteractions with gatekeeper Leu671. Additional substi-tuents, such as the –CH3 functional group, was welltolerated into the gatekeeper region. Further, the 4-ami-noaryl ring extended into the hydrophobic GXGXFGsite. The N,N-dimethyl aryl-sulfone faces the pocketsurrounded by Leu593 andVal601 and is positioned closeto the DFG site involved in polar interactions withAsp741 residue. The 2-position of the aryl-piperazinering moiety is exposed to the solvent accessible region,indicating that these positions should be able to accom-modate a variety of substitutions to improve the potencyand adjust physicochemical properties.

Despite the overall structural conservation, severalunique features of the active site can be exploited fordrug design. Unique to the TAM family of kinases is thepresence of nonpolar residues, such as Phe622/673 andPro621/672, located in the hinge region, which is adjacentto the gatekeeper site Leu620/671. These 2 unique resi-dues and their location aid in the process of designingspecific inhibitors. Sequence and structural similaritiesamong all 3 TAM family members suggest that it will be achallenge to design ATP-competitive small moleculeswith absolute specificity for a single family member.

Small molecule inhibitorsSmall molecule inhibitors of Axl and Mer have

emerged and are in various stages of drug development(Fig. 2B). Axl has received more focus from drug discov-ery programs, although inhibitors of Mer are also begin-ning to surface; however, no compounds specifically

designed to target Axl or Mer are in the clinical stagesof development. It is likely that some of the agentsdescribed below will enter clinical trials in the nearfuture.

According to published data, R-428 is the furthestadvanced Axl small molecule inhibitor in preclinical de-velopment (57). It is based on a trisubstituted triazole coreand exhibits potent activity against Axl kinase (IC50 ¼ 14nmol/L) in both biochemical and cell-based experi-ments. In a kinase selectivity panel, R-428 showed goodspecificity toward Axl kinase with some cross-reactivitywithMer, Tyro3, VEGFR family members, Ret, Tie2, andAbl kinases. In cell-based models, R-428 inhibited Gas6–stimulated phosphorylation of Axl and Akt (Ser473).R428 dose-dependently suppressed invasion of bothhuman MDA-MB-231 and murine 4T1 breast cancercells. In pharmacokinetic mouse studies, R-428 wasshown to have good plasma stability (half life ¼ 4–13hours), and pharmacologically relevant doses wereachievable by oral administration of the drug. Usingadditional animal models, R-428 was shown to inhibitbreast cancer metastasis and suppress angiogenesis,and these activities correlated with inhibition of Aktand Erk phosphorylation. Furthermore, R-428 showedgood synergistic activity with cisplatin in inhibitingliver and lung metastases in a breast cancer mousemodel. Overall, the data surrounding R-428 offer a goodrationale for its further development, particularly as anantimetastatic agent in breast cancer.

Other drug development groups have disclosed dis-covery programs focusing on Axl kinase, although thedetails surrounding these compounds are limited. A fewyears ago SGI-AXL-277, an Axl kinase inhibitor based ona pyrrolopyrimidine scaffold, was reported to be in leadoptimization and biological testing phases of preclinicaldevelopment (63). A series of Axl kinase inhibitors wereshown to inhibit proliferation of a diverse panel of celllines from hematologic malignancies (64).

CVO-102 is an inhibitor of Mer kinase in late preclinicaldevelopment, although only a small amount of informa-tion is available about this therapeutic agent (65). It hasbeen shown to have significant activity in preventingblood clotting and also shows promise as an anticanceragent, particularly in leukemias and lymphomas. Thegroup developing this compound has reported the gen-eration of some animal data to validate their approachand reportedly is continuing optimization of the leadagent.

Biological therapeuticsIn addition to these small molecule Axl andMer kinase

inhibitors, biological therapeutics are also in preclinicaldevelopment. Preclinical work was recently publishedusing a monoclonal antibody (YW327.6S2) recognizingboth human and murine forms of Axl (66). YW327.6S2was shown to block Axl function by downregulation ofthe expression of Axl as well as through inhibition ofGas 6 binding, leading to inactivation of Axl and its

Verma et al.





downstream signaling to Akt. As a single agent,YW327.6S2 showed significant activity against thegrowth of lung carcinoma cells (A549) inmouse xenograftmodels, along with the observed downregulation of Axlexpression and induction of apoptosis in these cells.YW327.6S2 also targeted tumor stromal cells by enhanc-ing the effects of an anti-VEGF antibody therapy ontumor vasculature, resulting in tumor stasis maintainedfor at least 4 weeks after treatment. Furthermore,YW327.6S2 sensitized wild-type EGFR lung carcinomacells to erlotinib treatment, suggesting Axl inhibitionmayenhance the efficacy of ERGR inhibitors in tumors that arerefractory to EGFR inhibition alone. Combination studiesfurther showed that YW327.6S2 synergized with carbo-platin/paclitaxel in the same lung cancer xenograft mod-el. In the MDA-MB-231 breast cancer xenograft model,YW327.6S2 showed activity as a single agent and incombination with an anti-VEGF therapy. In this model,significant effects were observed on reducing tumorvascular density, tumor-inflammatory response, andMDA-MB-231 metastases to the bone. The advantagesof a therapy based on a monoclonal antibody against theAxl receptor are clear, including the enhanced specificityand reduced off-target effects often achieved with biolog-ical therapies. An unexplored potential drawback associ-ated with this approach is that the Axl antibody may beprimarily binding to the sAxl in the bloodstream and,thereby, diminishing the effects of the antibody and re-quiring high doses of the antibody for effective treatment.An additional approach to targeting Gas 6/Axl signal-

ing would be to use recombinant sAxl as a sponge to soakup free circulating Gas 6 (67). The bound Gas 6 would be

unable to bind to the Axl receptor and initiate down-stream signaling consequences. This approach wouldpotentially have the advantage of using a biological agentwith high specificity and limited toxicity, but the Gas 6–independent effects of Axl activation are not well under-stood and are likely to play a role in tumorigenesis, asusing sAxl as a therapeutic agent would only inhibit theGas 6–dependent function of the Axl kinase receptor.

Conclusion

Axl andMer are novel RTKs and play a significant rolein pathogenesis and progression of human cancers. Theycan serve as important biomarkers for disease status andprognosis. Ongoing studies are using their unique struc-ture and role in cancer genesis and as a therapeutic targetfor drug development.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Jill Rhead, MA, FAMI, at the Intermountain Medical Centerfor assistance in generating the figures and illustrations.

Grant Support

This work was supported by funding from the Huntsman Cancer Foundationand NCI grant number P30 CA042014.

Received March 9, 2011; revised June 27, 2011; accepted July 2, 2011;published OnlineFirst September 20, 2011.

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2011;10:1763-1773. Published OnlineFirst September 20, 2011.Mol Cancer Ther Anupam Verma, Steven L. Warner, Hariprasad Vankayalapati, et al. Targeting Axl and Mer Kinases in Cancer

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